One third of the world's population is estimated to be infected with a dormant tuberculosis (TB) infection. Mycobacterium tuberculosis, the causative agent of TB, survives within this dormant state by engulfing host cell lipid bodies and then accumulating significant intracellular stores of lipids. Among the enzymes required for lipid metabolism and dormant infection in M. tuberculosis are a significant number of enzymes predicted to be hydrolases and specifically lipases. Recent work has established that inhibition of TB serine hydrolases can impede the growth and reactivation of dormant M. tuberculosis. TB hydrolases have thus become attractive therapeutic targets for the treatment of dormant TB infection, but the biological functions, enzymatic activity, and role in lipid metabolism of the majority of the 30 predicted hydrolases are unclear. The long-term goals are to characterize the biological functions of mycobacterial serine hydrolases in lipid metabolism and dormant TB infection and to determine if their enzymatic activities can be exploited to treat dormant TB infection. The objectives of this project are to develop novel imaging and antibacterial agents for the rapid characterization of the in vivo activity of mycobacterial serine hydrolases and to identify hydrolases essential to lipolysis. Synthesis and characterization of preliminary libraries of two classes of hydrolase substrates showed their synthesis to be reproducible, general, and rapid and the biochemical activity of hydrolases to differ based on hydrolase structure, activity, and biological function. Additionally, in Mycobacterium smegmatis, a fast-growing, non-infectious mycobacterial relative and model for lipid metabolism and energy mobilization, ester substrates have differential in vivo activity that is dependent on hydrolase activity. The majority of serine hydrolases and lipolytic processes from M. smegmatis are also conserved with M. tuberculosis. Additional preliminary bacterial hydrolase characterization led to the central hypothesis that serine hydrolases from mycobacteria have divergent substrate specificities that mimic their diverse lipid substrates and that these substrate preferences control the role of serine hydrolases in lipolysis and energy mobilization in mycobacterium. In Aim 1, novel chemical libraries of ester protected fluorogenic and antibacterial substrates will be synthesized and used to measure the global in vivo hydrolase activity of M. smegmatis. In Aim 2, zymography, mass spectrometry, mycobacterial genetics, fluorogenic hydrolase substrates, and heterologous bacterial expression will be combined to assign the substrate specificity profiles to individual hydrolases from M. smegmatis. In Aim 3, fluorogenic hydrolase substrates, hydrolase inactivation strains of M. smegmatis, and high-throughput fluorescence measurements will be used to establish the serine hydrolases directly involved in lipolysis and energy mobilization. With their essential roles in energy utilization and dormant TB survival, serine hydrolases involved in lipolysis could be used for the design of novel TB therapeutics treating dormant TB infection and for molecular diagnostics to visualize dormant TB infection.